Group Title: Genetic Vaccines and Therapy 2007, 5:13
Title: Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus
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Title: Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus
Series Title: Genetic Vaccines and Therapy 2007, 5:13
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Creator: Andino LM
Conlon TJ
Porvasnik SL
Boye SL
Hauswirth WW
Lewin AS
Publication Date: 39426
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Volume ID: VID00001
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Genetic Vaccines and Therapy


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Short paper


Rapid, widespread transduction of the murine myocardium using
self-complementary Adeno-associated virus
Lourdes M Andino', Thomas J Conlon2, Stacy L Porvasnik3, Sanford L Boye4,
William W Hauswirth4 and Alfred S Lewin*


Address: 'Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA, 2Powell Gene Therapy Center
University of Florida, Gainesville, FL, USA, 3Department of Pediatrics University of Florida, Gainesville, FL, USA and 4Department of
Ophthalmology, University of Florida, Gainesville, FL, USA
Email: Lourdes M Andino LAndino@ufl.edu; Thomas J Conlon conlon@gtc.ufl.edu; Stacy L Porvasnik porvas@ufl.edu;
Sanford L Boye sboye@ufl.edu; William W Hauswirth hauswrth@eye.ufl.edu; Alfred S Lewin* lewin@mgm.ufl.edu
* Corresponding author


Published: 10 December 2007
Genetic Voccines and Therapy 2007, 5:13 doi:10.1186/1479-0556-5-13


Received: I October 2007
Accepted: 10 December 2007


This article is available from: http://www.gvt-journal.com/content/5/l/l 3
2007 Andino et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the
incubation time needed to attain significant levels of gene expression is often too long for some
clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA,
eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene
expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency
of these two types of AAV vectors in the murine myocardium. Four day old CD- I mice were
injected with either of the two AAV constructs, both expressing GFP and packaged into the AAVI
capsid. The animals were held for 4, 6, I or 21 days, after which they were euthanized and their
hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR
quantification of AAV genome copies and for confocal microscopy. Although we observed similar
numbers of AAV genomes at each of the different time points present in both the scAAV and the
ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals
injected with the scAAV, while little or no expression was observed with the ssAAV constructs
until day I I. AAV transduction of murine myocardium is therefore significantly enhanced using
scAAV constructs.


Results and discussion
Adeno-associated virus has become an important tool for
gene transfer because of its lack of pathogenicity and its
ability to express passenger genes for long periods of time.
Although potentially safe as a gene therapy vector, this
virus exhibits an extended lag period before transgene
expression actually occurs. The reason for this delay in
expression is the binding of a cellular protein, FKBP52, to
the D-sequence within the inverted terminal repeats


(ITRs)[1]. Phosphorylated FKBP52 inhibits viral second
strand DNA synthesis, needed for transgene expression,
consequently leading to delayed transgene expression [1-
3]. As an avenue for bypassing this phenomenon, McCa-
rty et al. have introduced the use of a double-stranded
form ofAAV[4].

The typical single-stranded AAV (ssAAV) genome is
flanked by two, 145 bp ITRs. The 3' ITR serves as the rep-


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Genetic Vaccines and Therapy 2007, 5:13


location origin for the viral genome as well as a packaging
signal[5]. During replication, AAV genome dimers are
formed as replication intermediates. These dimers are
subsequently cleaved by AAV Rep proteins at the junction
of the ITR and the D sequence. Wang et al. discovered that
if one of the ITRs has a deleted D-sequence and terminal
resolution site (trs), cleavage by Rep cannot occur and
consequently, the dimers are not resolved into mono-
mers. Therefore, the double-stranded genomes are then
packaged as large hairpin DNA molecules[6].

Although the biology of these constructs has been studied,
testing of these constructs for gene delivery to animals is
in its early stages. In this report, we demonstrate the effi-
cacy of self complementary AAV (scAAV) in the murine
myocardium, a traditionally challenging organ to trans-
duce. Although different viruses have been employed for
myocardial gene transfer, each has its own limitations. For
example, adenovirus has been shown to be toxic and has
short-term expression[7], and lentiviral vectors stimulate
inflammatory responses[8]. Although adeno-associated
virus has a delay in onset and small packaging capacity, it
has been shown to direct gene expression for long periods
of time in the heart without any toxicity [9-11].

We compared the expression profile of both the ssAAV
and scAAV constructs in murine myocardia using sub-
xiphoid injections in 4 day old CD-1 mice (Charles Riv-
ers). The hearts were assessed using direct immunofluo-
rescence of the tissue 4, 6, 11, and 21 days post-injection.

The single stranded GFP-expressing AAV vector (ssAAV)
contained 4.3 kb of DNA surrounded by AAV-2 inverted
terminal repeats (ITR) (Figure 1A). Expression of GFP was
driven by the CMV enhancer-chicken B-actin promoter
(CBA) and contained the B-actin exon and the corre-
sponding 924 bp intron. Downstream of this was the
humanized GFP (GFPh) gene followed by a 1099 bp Neo-
mycin resistance cassette flanked with Sall cut sites. The
second plasmid vector used was very similar to ssAAV in
that it also expressed the GFPh gene under the control of
the CBA promoter (Figure 1B). This vector lacked the Neo-
mycin resistance cassette. Instead of the full length intron
and exon this construct had an intron of 202 bp contain-
ing the donor and acceptor sites necessary for splicing.
Additionally, as described by Wang et al., the 5' ITR had
the D-sequence and the trs site removed to prevent cleav-
age, and resolution of dimers by AAV Rep[6]. Therefore,
the total region to be packaged was 2,438 bp, making it
suitable to be packaged as a double-stranded or self-com-
plementary molecule (scAAV). The DNA from both vec-
tors was packaged according to previously reported
methods[ 12] into AAV1 capsids which are known to effi-
ciently transduce myocardial tissue[ 13].


PYF441 enhancer
Chicken p-actin promoter i 1 (2861) Sal 1(3960)
CMV i.e. enhancer Exonl SV40poly(A) HSV-k bGH poly(A)
TR GFPh neoR TR

I T I "
I ,


Chimeric infron


Chicken p-actin
CMV i.e. enhar


Figure I
Maps of scAAV and ssAAV. (A) A schematic representa-
tion of the single-stranded (ss) AAV genome containing a full-
length actin intron as well as a Neomycin resistance cassette.
This construct contains the CBA promoter driving the
expression of GFP. (B) A schematic representation of the
self-complementary (sc) AAV genome containing a mutated
5' inverted terminal repeat (ITR), an actin intron from which
722 nucleotides have been deleted, and no Neomycin resist-
ance cassette. This construct also contains the CBA pro-
moter driving the expression of GFP. Both of these
constructs contain the AAV2 ITRs and were packaged into
AAVI capsids.


Once the constructs were packaged, four day old CD-1
mice (Charles Rivers) were anesthetized on ice until
movement was barely visible, and injections into the car-
diac chamber were carried out as described by Zhang et
al[14]. Each animal was injected with 1.85 x 1011 total
particles of AAV1 containing either the ssAAV or the
scAAV expression cassettes. Three animals were injected
per group. Animals were returned to their dams for 4, 6,
11, or 21 days and then euthanized. Their hearts were
extracted and then cut into thirds representing the apical
region, the middle region and the base region of the heart.
The tissue was then fixed for 12 hours with 4% parafor-
maldehyde, rinsed in PBS and then allowed to equilibrate
in 20% sucrose for 12-24 hours. The next day, hearts were
frozen in OCT compound (Tissue-Tek). Five micron sec-
tions were cut in a cryostat and used for direct visualiza-
tion of GFP. Slides were then mounted using Vectashield


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Genetic Vaccines and Therapy 2007, 5:13


mounting media containing DAPI (Vector Labs). Two sec-
tions, each containing 75 Im of tissue, were reserved for
quantitative real-time PCR. Because of its high content of
adenine dinucleotides[15], cardiac tissue exhibits a high
level of autofluorescence and this background can
obscure the detection of GFP even when secondary anti-
bodies are employed[ 16]. Therefore, we chose to measure
the direct GFP signal generated using confocal microscopy
of the fixed tissues. This technique allowed us to almost
entirely eliminate background fluorescence seen with
conventional fluorescent microscopes and filters enabling
us to accurately represent the transduced cells. A Leica
confocal microscope was used to obtain fluorescent
images. Montages consisting of 2-4 fields of view per sec-
tion were created using Adobe Illustrator CS2 and Adobe
Photoshop CS2.

Four days post-injection, scAAV treated animals showed
widespread GFP expression throughout the myocardium
(Figure 2A) while ssAAV injected animals failed to reveal
any GFP expression (data not shown). Similarly, 6 days
post-injection GFP expression was detected throughout
the myocardium in scAAV injected mice (Figure 2B) while
the ssAAV injected animals exhibited no GFP expression
(data not shown). On the 11th day post-injection, a few
GFP positive cells were visible in the ssAAV treated hearts
(Figure 3A, top panels), while very high levels of GFP were
clearly visible in the apical, mid-section, and regions of
the base of scAAV treated hearts (Figure 3A, bottom pan-
els). At 21 days post-injection, GFP expressing cells were
apparent in the ssAAV infected hearts throughout the
myocardium (Figure 3B, top panels) although at a much
lower level than scAAV treated hearts (Figure 3B, bottom
panels) which had GFP expression throughout all three
regions of the myocardium.

One explanation for the increased expression of GFP
using scAAV is increased viral infection using that prepa-
ration relative to the ssAAV preparation. To determine
genome copy number in infected hearts, genomic DNA
(gDNA) was extracted from serial sections adjacent to the
ones used for immunofluorescence using the Qiagen
DNeasy tissue kit. One microgram of extracted gDNA was
used in all quantitative polymerase chain reactions.
Primer pairs were designed to the CBA promoter and
standard curves established by spike-in concentrations of
a plasmid DNA containing the same promoter. DNA sam-
ples were assayed in triplicate. The third replicate was
spiked with CBA DNA at a ratio of 100 copies/Gg of
gDNA. If at least 40 copies of the spike-in DNA were
detected, the DNA sample was considered acceptable for
reporting vector DNA copies. Results from these experi-
ments indicated that although there were differences in
the amount of GFP directly visualized using immunoflu-
orescence, the amount of AAV genome copies found in


the scAAV or ssAAV infected hearts were similar (Figure 4).
There was no statistical significance between the amounts
of vector genomes found in the scAAV infected heart ver-
sus an ssAAV infected heart. A gradual decline in AAV vec-
tor genomes observed over the time course of the
experiment could be attributed to the dilution of the viral
genomes as the heart grew within the developing pups as
well as due to natural cell death that occurs during devel-
opment.

We have described a novel self-complementary AAV vec-
tor that contains the chicken 3-actin promoter driving the
expression of GFP. Despite similar number of vector
genomes observed in hearts infected with either scAAV or
ssAAV, the scAAV transduced animals showed robust and
widespread GFP expression as early as 4 days post-injec-
tion, while even at 11 days, expression using ssAAV was
minimal. GFP expression could be seen all throughout the
heart from the base to the apex. The amount of GFP
expression increased over the time course of the experi-
ment in both of the scAAV and ssAAV transduced animals.
Nonetheless, the amount of GFP expression seen in the
scAAV injected animals was much higher than the ssAAV
injected animals at each time point.

Although this construct will not be suitable for packaging
large transgenes, this type of vector will be very useful for
the delivery of small transgenes and small molecules such
as ribozymes and shRNA molecules. Additionally, Wu et
al. have demonstrated that the packaging capacity of these
self-complementary vectors can be as large as 3.3 kb
which is more than what was originally expected[ 17]. If
efficient myocardial transduction can be demonstrated in
adult animals, these double-stranded AAV vectors might
ultimately prove useful in patients who need rapid expres-
sion of therapeutic genes.

Competing interests
We would like to disclose that William W. Hauswirth,
contributing author, and the University of Florida own
stock in the AGTC Corporation, which develops AAV tech-
nology.

Authors' contributions
LMA was involved in the conceptual design and acquisi-
tion of data. TJC assisted by performing quantitative real-
time PCR of vector genomes in cardiac tissue. SLP was
involved in tissue harvesting and processing. SLB gener-
ated the novel scAAV construct with the short chicken B-
actin promoter. WWH and ASL were the coordinators of
the project.







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Genetic Vaccines and Therapy 2007, 5:13


A Murne heart 4 days post-njecton wth sAAV
Murine heart 4 days post-injection with scAAV


B
Murine heart 6 days post-injection with scAAV


Figure 2
Rapid onset of gene expression using scAAV in the murine heart. CD-I mice (Charles Rivers) were injected 4 days
after birth with 1.85 x 1011 vector genomes (vg) of scAAV using previously established methods [14]. Animals were returned
to their dams for 4 days (A) or 6 days (B) at which time their hearts were harvested, cut into thirds representing the apical
region (apex), the mid-region (mid), or base of the heart (base). The tissues were fixed in 4% paraformaldehyde for 12 hours,
rinsed in PBS and allowed to equilibrate in 20% sucrose for 12-24 hours. The next day, the hearts were frozen in cryomolds
containing OCT compound (Tissue-Tek) to prepare for cryostat sectioning into 5 tm sections. Slides were mounted using
Vectashield mounting media containing DAPI (Vector Labs) to counter stain the nuclei. A Leica TCS SP2 AOBS Spectral Con-
focal Microscope with a I Ox objective was used to obtain fluorescent images. Staining was documented using the Leica Confo-
cal Software (LCS) Version 2.61. Sections with GFP expression can be seen in the top panels while merged images containing
GFP and DAPI signals can be seen in the lower panels. The bar represents 150 tm in the 6 day apex and 300 tm in all other
panels.




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Genetic Vaccines and Therapy 2007, 5:13


Murine heart 11 days post-injection with AAV


B
Murine heart 21 days post-injection with AAV


Figure 3
Increased gene expression with scAAV after longer intervals. CD-I mice (Charles Rivers) injected 4 days after birth
with 1.85 x 1011 vector genomes (vg) of either scAAV or ssAAV using sub-xiphoid injections [14]. The animals were returned
to their dams for I I days (A) or 21 days (B) and then processed as described in Figure 2. Representative sections with GFP flu-
orescence are pictured. The bar represents 150 im in the 21 day ssAAV-apex and 300 im in all other panels.




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Genetic Vaccines and Therapy 2007, 5:13


4 days 6 days 11 days 21 days


Figure 4
Similar copy numbers of ssAAV and scAAV in
infected hearts. Serial sections of tissues used for micros-
copy were collected for real-time PCR analysis of AAV vec-
tor genomes. Genomic DNA (gDNA) was extracted from
the tissues using a Qiagen DNeasy tissue kit according to the
manufacturer's protocol. One microgram of extracted
gDNA was used in all quantitative PCR reactions. The PCR
conditions were 50 cycles of 94.8oC for 40 s, 37.8oC for 2
min, 55.8oC for 4 min, and 68.8oC for 30 sec. DNA samples
were assayed in triplicate. The third replicate was spiked
with CBA DNA at a ratio of 100 copies/ig of gDNA. If at
least 40 copies of the spike-in DNA were detected, the DNA
sample was considered acceptable for reporting vector DNA
copies. Data were averaged by group and plotted with stand-
ard deviation for comparisons. No statistically significant dif-
ferences were measured between any of the groups of
scAAV and ssAAV treated animals.



Acknowledgements
The authors' would like to thank the Powell Gene Therapy Center for the
assistance in quantitating the AAV vector genomes in the murine cardiac
tissue samples. We acknowledge NIH grants EY 13729, EY I 1123, NS36302,
EY08571, and grants from the Macular Vision Research Foundation, Foun-
dation Fighting Blindness, Juvenile Diabetes Research Foundation and
Research to Prevent Blindness, Inc. for partial support of this work. Lour-
des M. Andino was the recipient of an American Heart Association predoc-
toral fellowship.

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